Abstract 14996: Single Cell Rnaseq Analysis Reveals a Two-Step Mechanism for the Reprogramming of Ventricular Myocytes to Pacemaker Cells by Tbx18

Abstract

Background: We have demonstrated that an embryonic transcription factor, TBX18, suffices to reprogram ventricular myocytes (VMs) to induced pacemaker cells (iPMs). Here, we sought to gain a mechanistic understanding of the reprogramming process using single cell (sc) RNAseq analysis. Methods: Cells from the ventricles of the neonatal rat heart were plated as monolayers, which consisted of both cardiomyocytes (CMs) and nonmyocytes, and were transduced with Adeno-GFP and Adeno-TBX18 in vitro. scRNAseq was performed at days (d) 3, 6, and 14. Result: TBX18- CMs exhibited repression of chamber CM genes at d3, including those involved in cardiac muscle contraction and action potential. Markers of CM dedifferentiation ( Acta2 and Dab2 ) were upregulated, while markers of chamber CM differentiation ( Mef2a and Rbm20 ) were downregulated. Nodal pacemaker cell gene expression followed the CM dedifferentiation in TBX18-CMs, with the proportion of Hcn4 + CMs increasing gradually from 20.9% (d3) to 34.0% (d14), accompanied by increased expression of Tbx3 and Tbx18 . The proportion of iPMs ( Hcn4 + , Gja1 low , Nkx2-5 low , Tnni3 high , and Actn2 high ) in TBX18-CMs also increased from 3.6% at d3 to 6.4% at d14. Both TBX18-CMs and GFP-CMs exhibited a subpopulation of Acta2 high dedifferentiated CMs (deCMs), which were represented higher in TBX18-CMs compared to control. Among TBX18-CM subpopulations, the proportion of iPMs was the highest in deCMs (6.4% at d3 and 7.3% at d14). TBX18-deCMs were distinct from other TBX18-CM subpopulations in that extracellular matrix organization and Tgfβ signaling were enriched as well as higher expression of SP1 transcription factor which directly activates Hcn4 channel expression. Conclusion: Dedifferentiation and loss of CM function precedes nodal pacemaker cell gain of function during TBX18-induced somatic reprogramming of VMs to iPMs. Tgfβ signaling appears to figure prominently during this process.